FUNCTIONALIZED POLYMERIC SCAFFOLDS FOR NEURAL TISSUE ENGINEERING

Abstract

Much effort has to be taken during devising and fabricating biocompatible and functional scaffolds for nervous system regeneration. Considering the very demanding nature of neurons, it becomes very challenging to tackle the issues like biocompatibility, mechanical stability and electrical cues. Apart from these it is needed to consider geometrical cues. At present, mainly polymeric blends are being used for fabricating scaffolds. Out of these, some polymers are conducting in nature but their conductivity is quite low. So for electrical cues, we need conductive nanofiller. Additionally, polymers are also having quite low mechanical stability. Carbon nanofiller are good choice to enhance conductivity and mechanical properties of polymers. Till now such patterned carbon nanofillers for functionalized neural scaffolds are being grown on top surfaces of substrates like glass, mica and silicon via CVD on lithographically synthesized patterns. Such structures do not offer enough adhesion strength of carbon nanofillers to the scaffold surface, posing a high risk of in-vivo release of the nanostructures when implanted. Once uprooted from the surface and taken away by circulatory system carbon nanofillers are not able to offer the geometrical and electrical cues through the scaffold in long term. Apart from this issue, there is another serious problem in using such carbon nanofiller grown structures as neural scaffold. Carbon nanofiller growth through chemical routes on substrates requires very high temperature, which polymers cannot withstand. Thus, most of these studies have used silicon or metallic materials as substrate. However, these hard and stiff substrates cannot be used in-vivo as scaffold, considering the soft structure of the neural tissues. As it shows, these scaffolds are not a real solution, which can be used for nerve tissue engineering. Thus, the ideal solution at this point is to have a soft base material, like polymer, for neural scaffold, with carbon nanofillers attached strongly or embedded in the structure following some pattern, i.e., alignment. This can only be achieved by synthesizing a polymer based composite, in which the carbon nanofillers can be aligned as per the requirement of the nerve tissue it is going to replace. However, it is not an easy task to align the carbon nanofillers in any composite. Therefore, the critical step was to identify and develop a suitable fabrication to get carbon nanofiller aligned in polymer matrix, suitable for neural tissue replacement. Once successfully fabricated, this scaffold should be able to offer effective electrical and geometrical cues ideal for nerve tissue engineering. Thus, the aim of this research was to develop a modified scaffold that can solve biological, mechanical, electrical and topographical cues of neural tissue engineering. The scaffolds were prepared via 2 methods: solution casting and electrospinning and further characterized for morphology, mechanical strength, electrical conductivity, neuro cytocompatibility and electrophysiology by calcium imaging. “ Before fabricating the biocompatible scaffold for neural tissue engineering, initially two different methods of alignment i.e. alternating voltage assisted and current-assisted alignment were compared on multi-walled carbon nanotubes (MWCNT) reinforced polyvinylidene fluoride (PVDF) composite. The morphological and alignment of MWCNTs inside polymer matrix were assessed by SEM. The mechanical properties and electrical conductivities are also characterized. MWCNTs are“aligned with the application of alternating voltage as well as pulsed current. The voltage-assisted films have shown good dispersion and alignment of CNTs inside PVDF matrix. On the other hand, the current-assisted alignment led to the formation of shortest, continuous path for the flow of electrons and hence, resulted in formation of highly anisotropic conductive film. Directly current passage assisted alignment of CNTs in the composite film records an impressive 361% improvement in conductivity in the direction parallel to the alignment as compared to the structure with randomly aligned CNT. At the same time, the composite in transverse direction to the alignment was totally insulating, indicating the efficiency of alignment. With the addition of only 0.5 wt% CNTs to PVDF matrix, film shows improvement of elastic modulus and tensile strength by 181% and 148%, respectively, as compared to pure PVDF film. However, the films behaved mostly isotropic in terms of mechanical properties, showing improvement in all the directions with CNT reinforcement.”After getting the versatility of effects of voltage assisted alignment, similar process was conducted on biocompatible chitosan scaffolds. Uniform distribution and alignment of 0.5 wt.% ,MWCNT in the chitosan matrix, good interfacial bonding and aligned network of CNT helps in improving the elastic modulus, yield strength and ultimate tensile strength by 12.7%, 21.92% and 11.2%, respectively, as compared to the random MWCNT-chitosan scaffold. Alignment of MWCNT introduces highly anisotropic electrical conductivity (100000 times higher) in alignment direction, as compared to the transverse direction of the scaffold. Interactions of HT-22 hippocampal neurons with MWCNT-chitosan matrix prove them to be highly biocompatible with a notable increase in viability. In addition, 50-60% neurons are found to be aligned in the MWCNT alignment direction of the scaffold”Further, taking the consideration of results from above results, 0.5 wt.% GNP were also aligned inside chitosan matrix by an alternating bias electric field. The suitability of anisotropic conductivity was evaluated by culturing HT-22 neural cells on the scaffold which revealed no directional growth is accomplished for the random as well as aligned scaffolds. The effect of the difference in morphology of CNT and GNP was also evaluated for HT-22 neurons. The hybrid v MWCNT/GNP/chitosan scaffolds were fabricated to assess the difference in behavior of HT-22 neurons. Electrical conductivities of all the hybrid scaffolds are found to be in between that of MWCNT/chitosan scaffold (highest-conductivity) and GNP/chitosan scaffold (lowest-conductivity). While, hybrid scaffolds show improvement in elastic modulus and ultimate tensile strength over MWCNT/chitosan and GNP/chitosan scaffolds. The protein adsorption isotherms of bovine serum albumin (BSA) show greater equilibrium constant (Keq) on GNP/chitosan composites as compared to MWCNT/chitosan composites, proving more potential for cell adhesion in the former. Interactions of HT-22 hippocampal neurons with MWCNT/chitosan, GNP/chitosan and various MWCNT/GNP hybrid chitosan matrices prove cytocompatibility. The neurons acquire elongated geometry on the MWCNT/chitosan scaffold, while GNP reinforcement drives the neurons to spread cellular processes radially. Finally, the fabrication and comprehensive cytocompatibility assessment of 3D structured electrospun nano-fibrous multiwalled carbon nanotube (MWCNT) reinforced polymer scaffolds was done. Scaffolds prepared via electrospinning possess high porosity and nano- and micro-scale interconnected pores. The electrospun scaffolds were also characterized for their tensile strength and electrical conductivity. The in-vitro 3D neuronal circuits of primary hippocampal neurons instructed 3D growth. The 3D systems allowed exploring the emerging electrophysiological activity, in terms of calcium signals. The neuronal activity emerges as a function of the interplay between the 3D electrically conductive architectures and network dynamics. The improvement in functional organization and synchronization was also reported in small neuronal assemblies. Reinforcing the carbon nanotubes in the electrospun scaffolds altered synaptic activity remarkably, therefore supporting nanomaterial/cell interfacing in 3D growth support. The 3D system represents a simple and reliable neural construct, having ability to develop the complexity of current neural tissue culture models.